Antiviral Properties of Silver Nanoparticles on a Magnetic HybridColloid

SungJun Park,a Hye Hun Park,b Sung Yeon Kim,a Su Jung Kim,a Kyoungja Woo,b GwangPyo Koa,c

Department of Environmental Health, Graduate School of Public Health, Seoul National University, Seoul, Republic of Koreaa; Molecular Recognition Research Center,Korea Institute of Science and Technology, Seoul, Republic of Koreab; Bio-MAX Institute, Seoul National University, Seoul, Republic of Koreac

Silver nanoparticles (AgNPs) are considered to be a potentially useful tool for controlling various pathogens. However, there areconcerns about the release of AgNPs into environmental media, as they may generate adverse human health and ecological ef-fects. In this study, we developed and evaluated a novel micrometer-sized magnetic hybrid colloid (MHC) decorated with vari-ously sized AgNPs (AgNP-MHCs). After being applied for disinfection, these particles can be easily recovered from environmen-tal media using their magnetic properties and remain effective for inactivating viral pathogens. We evaluated the efficacy ofAgNP-MHCs for inactivating bacteriophage �X174, murine norovirus (MNV), and adenovirus serotype 2 (AdV2). These targetviruses were exposed to AgNP-MHCs for 1, 3, and 6 h at 25°C and then analyzed by plaque assay and real-time TaqMan PCR. TheAgNP-MHCs were exposed to a wide range of pH levels and to tap and surface water to assess their antiviral effects under differ-ent environmental conditions. Among the three types of AgNP-MHCs tested, Ag30-MHCs displayed the highest efficacy for inac-tivating the viruses. The �X174 and MNV were reduced by more than 2 log10 after exposure to 4.6 � 109 Ag30-MHCs/ml for 1 h.These results indicated that the AgNP-MHCs could be used to inactivate viral pathogens with minimum chance of potential re-lease into environment.

With recent advances in nanotechnology, nanoparticles havebeen receiving increased attention worldwide in the fields of

biotechnology, medicine, and public health (1, 2). Owing to theirhigh surface-to-volume ratio, nano-sized materials, typicallyranging from 10 to 500 nm, have unique physicochemical prop-erties compared with those of larger materials (1). The shape andsize of nanomaterials can be controlled, and specific functionalgroups can be conjugated on their surfaces to enable interactionswith certain proteins or intracellular uptake (3–5).

Silver nanoparticles (AgNPs) have been widely studied as anantimicrobial agent (6). Silver is used in the creation of fine cut-lery, for ornamentation, and in therapeutic agents. Silver com-pounds such as silver sulfadiazine and certain salts have been usedas wound care products and as treatments for infectious diseasesdue to their antimicrobial properties (6, 7). Recent studies haverevealed that AgNPs are very effective for inactivating varioustypes of bacteria and viruses (8–11). AgNPs and Ag� ions releasedfrom AgNPs interact directly with phosphorus- or sulfur-contain-ing biomolecules, including DNA, RNA, and proteins (12–14).They have also been shown to generate reactive oxygen species(ROS), causing membrane damage in microorganisms (15). Thesize, shape, and concentration of AgNPs are also important factorsthat affect their antimicrobial capabilities (8, 10, 13, 16, 17).

Previous studies have also highlighted several problems whenAgNPs are used for controlling pathogens in a water environment.First, existing studies on the effectiveness of AgNPs for inactivat-ing viral pathogens in water are limited. In addition, monodis-persed AgNPs are typically subject to particle-particle aggregationbecause of their small size and large surface area, and these aggre-gates reduce the effectiveness of AgNPs against microbial patho-gens (7). Finally, AgNPs have been shown to have various cyto-toxic effects (5, 18–20), and the release of AgNPs into a waterenvironment could result in human health and ecological prob-lems.

Recently, we developed a novel micrometer-sized magnetic hy-

brid colloid (MHC) decorated with AgNPs of various sizes (21,22). The MHC core can be used to recover the AgNP compositesfrom the environment. We evaluated the antiviral efficacy of thesesilver nanoparticles on MHCs (AgNP-MHCs) using bacterio-phage �X174, murine norovirus (MNV), and adenovirus underdifferent environmental conditions.

MATERIALS AND METHODSSynthesis of Ag07-MHC, Ag15-MHC, and Ag30-MHC. All of the mag-netic hybrid colloids decorated with AgNPs of various sizes (AgNP-MHCs) were synthesized by the Molecular Recognition Research Centerof the Korea Institute of Science and Technology (KIST), Seoul, SouthKorea. Materials were used as purchased and as reported in our previousstudy (21). Ag07-MHC, Ag15-MHC, and Ag30-MHC were synthesizedusing the same procedure as reported in our previous studies (21, 22). Anaminopropyl-functionalized Fe3O4-SiO2 core-shell magnetic hybrid col-loid (AP-MHC) stock solution containing 3.7 � 1010 AP-MHCs/ml wasprepared and used as required.

Briefly, silver nanoparticles of 7, 15, and 30 nm were grown on theoutermost surface of a MHC through a seeding, coalescing, and growingstrategy (21, 22). Five milliliters of AP-MHC was mixed with 25 ml of Auseed solution (22). The Au-seeded MHC was collected by magnetic de-cantation and dispersed in 5 ml of deionized water (DW). To grow 7-nm(or 15-nm) AgNPs on the surface of the MHC, a mixture of 40 ml (or 80ml) of AgNO3 (0.01% [wt/vol] in water) and 0.004 ml (or 0.008 ml) ofNH4OH (30% in water) were prepared with stirring. After 5 min, 0.03 mlof formaldehyde (37% in water) was added. The mixture was stirred witha mechanical stirrer for 30 min and allowed to sit for 1.5 h. The solid was

Received 16 October 2013 Accepted 27 January 2014

Published ahead of print 31 January 2014

Editor: J. L. Schottel

Address correspondence to GwangPyo Ko, gko@snu.ac.kr.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.03427-13

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purified by washing with DW three times using magnetic decantation anddispersed in 20 ml of DW, resulting in a solution containing 9.2 � 109

Ag07-MHC (or Ag15-MHC) particles/ml of solution.To synthesize Ag30-MHC, the pH-adjusted (pH �4) AP-MHC solu-

tion (5 ml) was slowly added to 25 ml of the Ag seed solution (21). TheAg-seeded MHC was collected by magnetic decantation and dispersed in5 ml of DW. This solution was added to a mixture of AgNO3 (0.02 g) andNH4OH (30%, 0.04 ml) in 200 ml of DW and stirred for 10 min with amechanical stirrer in an ice bath. After 0.05 ml of formaldehyde wasadded, the solution was stirred for 30 min and left for 1.5 h withoutperturbation. The resulting Ag30-MHC was purified by washing with DWthree times using magnetic decantation and dispersed in 20 ml of DW(9.2 � 109 particles/ml). The completed AgNP-MHC solutions werestored at room temperature (25°C) in the dark.

The AgNP-MHCs were characterized by transmission electron mi-croscopy (TEM) using a CM30 transmission electron microscope (PhilipsInc., USA) equipped with an energy-dispersive spectrometer and by scan-ning electron microscopy (SEM) using an XL30 environmental scanningelectron microscope (ESEM) (FEI Co., USA). Images were captured at theAdvanced Analysis Center of KIST (Seoul, South Korea), as in our previ-ous studies (21).

Preparation of target virus stock. Bacteriophages MS2 (ATCC 15597-B1) and �X174 (ATCC 13706-B1) were propagated using the single-agar-layer technique (23–25). Initially, both bacteriophages were culturedovernight at 37°C with Escherichia coli C3000 (ATCC 15597) as the hostbacteria. The phages were then washed using phosphate-buffered saline(PBS) and purified from E. coli lysates as described previously with somemodifications (26). Briefly, an equal volume of chloroform was added tothe lysates, followed by centrifugation at 5,000 � g for 20 min at 4°C. Thesupernatant was recovered as the phage stock and stored at �80°C untiluse.

MNV was propagated in RAW 264.7 cells as described previously withsome modifications (26). RAW 264.7 cells were cultured in Dulbecco’smodified Eagle’s medium (DMEM) (Gibco, USA) containing 10% fetalbovine serum (Gibco), 10 mM HEPES (Gibco), 10 mM sodium bicar-bonate (Gibco), 10 mM nonessential amino acids (Gibco), and 50 �g/�lgentamicin reagent (Gibco). MNV was then inoculated onto a monolayerof RAW 264.7 cells in a sterilized flask and cultivated for 3 to 4 days.Infected cells were subjected to three cycles of freezing and thawing tocause cell membrane damage, allowing the easy release of MNV inside theinfected cells. An equal volume of chloroform was added to the cell lysatesand mixed, followed by centrifugation at 5,000 � g for 20 min at 4°C.Centrifugal ultrafiltration using Amicon Ultra-15 tubes (Millipore, USA)was used to concentrate the titer of MNV, and the virus stock was stored at�80°C until use.

Adenovirus serotype 2 (AdV2) was propagated in A549 cells as de-scribed previously with some modifications (27). A549 cells were culturedin RPMI 1640 medium (Gibco) containing 10% fetal bovine serum, 10mM HEPES, 10 mM sodium bicarbonate, 10 mM nonessential aminoacids, and 50 �g/�l gentamicin reagent. AdV2 was then inoculated ontothe cultivated A549 cells, and the infected cells were subjected to threecycles of freezing and thawing. An equal volume of chloroform was addedto the cell lysates and mixed, followed by centrifugation at 5,000 � g for 20

min at 4°C. Centrifugal ultrafiltration using Amicon Ultra-15 tubes (Mil-lipore, USA) was used to concentrate the titer of AdV2, and the virus stockwas stored at �80°C until use.

Antiviral properties of AgNP-MHCs. To assess the antiviral proper-ties of AgNP-MHCs, each type of AgNP-MHC was used at three differentconcentrations (4.6 � 107, 4.6 � 108, and 4.6 � 109 particles/ml). Briefly,bacteriophage �X174, MNV, and AdV2 were treated with Ag07-MHCs,Ag15-MHCs, and Ag30-MHCs in a shaking incubator at 150 rpm for 1 hat 25°C. As a control, OH-MHCs with no AgNPs were used at 4.6 � 109

particles/ml. In addition, the viruses were exposed to each AgNP-MHC at4.6 � 109 particles/ml for 1, 3, and 6 h at 25°C.

After exposure to the particles, plaque assays and real-time TaqManPCR (RT-PCR) assays were used to measure the efficacy of AgNP-MHCsfor inactivating the tested viruses. For the RT-PCR assays, the viral ge-nomes of MNV and AdV2 were extracted using a QIAamp MinElute virusspin kit (Qiagen, USA). RT-PCR for MNV was performed using a Ag-Path-ID one-step RT-PCR kit (Ambion, USA) as described previously(28). Briefly, the nucleic acids from MNV were reverse transcribed at 48°Cfor 30 min and denatured initially at 95°C for 10 min, followed by 45cycles of 95°C for 15 s and 60°C for 1 min using a 7300 real-time PCRsystem (Applied Biosystems, USA). For RT-PCR of AdV2, TaqMan uni-versal PCR master mix (Applied Biosystems) was used. The AdV2 nucleicacids were reverse transcribed at 50°C for 2 min and denatured at 95°C for10 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. Thesequences of the primers and TaqMan probe used for RT-PCR are given inTable 1 (26, 27).

Effects of pH on the efficacy of AgNP-MHCs. To characterize theeffects of pH on AgNP-MHCs, 4.6 � 109 particles/ml of Ag30-MHCs wereexposed to acidic (pH 2.0 and 5.0) or alkaline (pH 9.0 and 12.0) DWsolutions in a shaking incubator at 150 rpm for 10 min at 25°C as de-scribed previously (29). The pH values were measured using an Orion3-Star benchtop pH meter (Thermo Fisher Scientific, USA). After acid oralkali treatment, the DW was neutralized to pH 7.6. The Ag30-MHCswere recovered from the solution using a strong magnet and suspended inDW at their initial concentration. The Ag30-MHCs exposed to pH 2.0,7.0, and 12.0 were examined using a Libra energy-filtering transmissionelectron microscope (Carl Zeiss Co. Ltd., South Korea) at the NationalInstrumentation Center for Environmental Management (NICEM),Seoul National University. Approximately 1 � 106 PFU/ml of both bac-teriophage MS2 and �X174 were mixed with Ag30-MHCs with and with-out previous exposure to acid or alkali. The mixtures were placed in ashaking incubator at 150 rpm for 1 h at 25°C, and the efficacy of virusinactivation was measured using the single-agar-layer technique de-scribed previously (24).

Antiviral activity of AgNP-MHCs in tap and surface water. Surfacewater was collected from the Han River in Seoul, South Korea, in July2011. All water samples were collected in 1-liter sterilized bottles andstored at 4°C until use. Physicochemical analyses of the sampled surfacewater and tap water were performed by a commercial company (Wendi-Bio Inc., Seongnam, South Korea), using the water analysis techniquesapplicable to the Korean standards for drinking water quality. Half of thecollected surface water samples were filtered through a 0.22-�m-pore-sizesyringe filter (Millex; Millipore). The concentrations of microorganisms

TABLE 1 Oligonucleotide primer and probe sequences for TaqMan real-time RT-PCR assay of AdV2 and MNV

Virus Primer or probe Name (position, 5=¡3=) Sequence Reference

AdV2 Forward primer JHKXF (18891–19910) 5=-GGA CGC CTC GGA GTA CCT GA-3= 27Reverse primer JHKXR (19025–19007) 5=-CGC TGI GAC CIG TCT GTG G-3=Probe JHKXP (18939–18960) 5=-FAM-CAC CGA TAC GTA CTT CAG CCT G-MGB-3=

MNV Forward primer MNV1 F (5614–5630) 5=-ACG CCA CTC CGC ACA AA-3= 26Reverse primer MNV1 R (5649–5657) 5=-GCG GCC AGA GAC CAC AAA-3=Probe MNV1 P (5632–5646) 5=-VIC-AGC CCG GGT GAT GAG-MGB-3=

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were measured by cultivation prior to an assay using the single-agar-layertechnique (24).

Approximately 4.6 � 109 Ag30-MHCs/ml were added to the raw andfiltered surface water samples and tap water samples in a shaking incuba-tor at 150 rpm for 10 min at 25°C. Approximately 1 � 106 PFU/ml of MS2and �X174 were then added to the water-treated Ag30-MHCs in a shakingincubator at 150 rpm for 1 h at 25°C, and virus inactivation was deter-mined using the single-agar-layer technique.

Statistical analysis. The data are expressed as means � standard de-viations (SD) from at least three independent experiments. When appro-priate, the data were analyzed with one-way analysis of variance(ANOVA) or Kruskal-Wallis one-way ANOVA and Dunnett’s test formultiple comparisons. P values of 0.05 were considered to indicate sta-tistical significance. SPSS for Windows (ver. 19.0; IBM, USA) and Sigma-plot for Windows (ver. 12.0; Systat software Inc., USA) were used forstatistical analyses.

RESULTSCharacterization of AgNP-MHCs. AgNP-MHCs were coatedwith AgNPs of different sizes. The properties of the AgNP-MHCsare presented in Table 2. Given the concentration of AgNP-MHCs(9.2 � 109 particles/ml), the number of AgNPs per MHC and thesilver concentration varied. As a result, AgNPs with the largestparticle size (Ag30-MHCs) had the highest concentration of silverper ml (400 ppm) and the lowest number of AgNPs per MHC (290particles) compared with Ag07-MHCs (57.5 ppm and 2,600 par-ticles). Our data indicate that Ag15-MHCs had the highest totalsurface area of AgNPs per MHC (1.1 �m2) and the highest surfacecoverage by AgNPs per MHC (25%).

Antiviral effects of AgNP-MHCs. The antiviral effects of theAgNP-MHCs with various particle sizes and at different concen-trations were measured by a plaque assay using bacteriophage�X174, MNV, and AdV2 (Fig. 1). The control treatment withOH-MHCs had no significant antiviral effect. In contrast, theAgNP-MHCs produced significant antiviral effects against bacte-riophage �X174 and MNV but not against AdV2 (Fig. 1 and 2). Alonger incubation time with the Ag30-MHCs produced a signifi-cantly greater reduction in bacteriophage �X174 and MNV.Compared with the other two AgNP-MHCs, Ag30-MHCs exhib-ited significantly greater antiviral effects for bacteriophage �X174and MNV (P 0.05). An approximate 6-log10 reduction of MNVand 4-log10 reduction of �X174 occurred after exposure to 4.6 �109 Ag30-MHCs/ml for 6 h, as determined using a plaque assay. Incontrast, when inactivation of MNV was analyzed by RT-PCR,only a 2-log10 reduction was observed. AdV2 had the highest re-sistance to AgNP-MHCs and did not display any significant inac-tivation regardless of the type of AgNP-MHCs used. The plaque

assay and RT-PCR results for AdV2 were not significantly differ-ent (Fig. 2d and e).

Effect of exposure of AgNP-MHCs to various pH conditionsprior to use. Figure 3 shows the antiviral efficacies of AgNP-MHCs that had been exposed to different pH conditions. Expo-sure to extremely acidic conditions (pH 2.0) significantly reducedthe antiviral capabilities of Ag30-MHCs. After 10 min of exposureto pH 2.0 conditions, the Ag30-MHCs produced only a 0.1-log10

reduction of bacteriophage MS2 and a 0.8-log10 reduction of bac-teriophage �X174, indicating antiviral efficacy significantly lowerthan that of untreated Ag30-MHCs (P 0.05). Exposure to astrongly alkaline condition (pH 12.0) also decreased antiviral ef-ficacy.

Antiviral effects of AgNP-MHCs in tap and surface water.The antiviral efficacy of AgNP-MHCs against bacteriophages MS2and �X174 in environmental water samples and in tap water sam-ples was investigated (Fig. 4). Various physicochemical character-istics of the river water samples are summarized in Table 3. Theefficacy of AgNP-MHCs against �X174 was maintained in thewater samples, but the antiviral efficacy against MS2 was lower inthe water samples than in DW.

DISCUSSION

This study demonstrated that AgNP-MHCs are effective for inac-tivating bacteriophages and MNV, a surrogate for human norovi-rus, in water. In addition, AgNP-MHCs can be easily recoveredwith a magnet, effectively preventing the release of potentiallytoxic AgNPs into the environment. A number of previous studieshave shown that the concentration and particle size of AgNPs arecritical factors for inactivating targeted microorganism (8, 16, 17).The antimicrobial effects of AgNPs also depend on the type ofmicroorganism. The efficacy of AgNP-MHCs for inactivating�X174 followed a dose-response relationship. Among the AgNP-MHCs tested, Ag30-MHCs had a higher efficacy for inactivating�X174 and MNV. For MNV, only Ag30-MHCs displayed antivi-ral activity, with the other AgNP-MHCs not generating any sig-nificant inactivation of MNV. None of the AgNP-MHCs had anysignificant antiviral activity against AdV2.

In addition to particle size, the concentration of silver in theAgNP-MHCs was also important. The concentration of silver ap-peared to determine the efficacy of the antiviral effects of AgNP-MHCs. The silver concentrations in solutions of Ag07-MHCs andAg30-MHCs at 4.6 � 109 particles/ml were 28.75 ppm and 200ppm, respectively, and correlated with the level of antiviral activ-ity. Table 2 summarizes the silver concentrations and surface areasof the AgNP-MHCs tested. Ag07-MHCs displayed the lowest an-tiviral activity and had the lowest silver concentration and surfacearea, suggesting that these properties are related to the antiviralactivity of AgNP-MHCs.

Our previous study indicated that the major antimicrobialmechanisms of AgNP-MHCs are the chemical abstraction ofMg2� or Ca2� ions from microbial membranes, the creation ofcomplexes with thiol groups located at the membranes, and thegeneration of reactive oxygen species (ROS) (21). Because AgNP-MHCs have a relatively large particle size (�500 nm), it is unlikelythat they can penetrate a viral capsid. Instead, AgNP-MHCs ap-pear to interact with viral surface proteins. AgNPs on the compos-ites tend to bind thiol group-containing biomolecules embeddedin the coat proteins of viruses. Therefore, the biochemical prop-erties of viral capsid proteins are important for determining their

TABLE 2 AgNP composite samples (AgNP-MHCs) used in this studya

Type of AgNPSilver concn(ppm)b

No. of AgNPsper MHC

Total surfacearea ofAgNPs perMHC(�m2)c

Surface coverageby AgNPs perMHC (%)

Ag07-MHC 57.5 2,600 0.41 8.8Ag15-MHC 275 1,600 1.1 25Ag30-MHC 400 290 0.81 18a Cited in our previous study (21). AgNP-MHCs were used at 9.2 � 109 particles/ml.b The Ag concentration was obtained from the Advanced Analysis Center of KIST usingatomic absorption spectrometer (AAS) and inductively coupled plasma (ICP) analysis.c Calculated by the following formula: surface area of one AgNP (4r2) � number ofAgNPs/MHC.

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FIG 1 Antiviral effects of AgNP-MHCs at various concentrations against bacteriophage �X174 (a), MNV (b), and AdV2 (c). Target viruses were treated withdifferent concentrations of AgNP-MHCs, and with OH-MHCs (4.6 � 109 particles/ml) as a control, in a shaking incubator (150 rpm, 1 h, 25°C). The plaque assaymethod was used to measure surviving viruses. Values are means � standard deviations (SD) from three independent experiments. Asterisks indicate signifi-cantly different values (P 0.05 by one-way ANOVA with Dunnett’s test).

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FIG 2 Antiviral effects of AgNP-MHCs after various reaction times. (a) Bacteriophage �X174; (b) MNV, plaque assay; (c) MNV, RT-PCR; (d) AdV2, plaque assay; (e)AdV2, RT-PCR. Target viruses was treated with AgNP-MHCs or OH-MHCs (control) at 4.6 � 109 particles/ml in a shaking incubator (25°C, 150 rpm) for 1, 3, and 6h. Surviving viruses were measured by plaque assay and RT-PCR. The results are expressed as means � standard deviations (SD) from three independent experiments.

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susceptibility to AgNP-MHCs. Figure 1 shows the different sus-ceptibilities of the viruses to the effects of AgNP-MHCs. The bac-teriophages �X174 and MNV were susceptible to AgNP-MHCs,but AdV2 was resistant. The high resistance level of AdV2 is likelyto be associated with its size and structure. Adenoviruses range insize from 70 to 100 nm (30), making them much larger than�X174 (27 to 33 nm) and MNV (28 to 35 nm) (31, 32). In additionto their large size, adenoviruses have double-stranded DNA, un-like other viruses, and are resistant to various environmentalstresses such as heat and UV radiation (33, 34). Our previousstudy reported that almost a 3-log10 reduction of MS2 occurred

with Ag30-MHCs within 6 h (21). MS2 and �X174 have similarsizes with different types of nucleic acid (RNA or DNA) but havesimilar rates of inactivation by Ag30-MHCs. Therefore, the natureof the nucleic acid does not appear to be the major factor forresistance to AgNP-MHCs. Instead, the size and shape of viralparticle appeared to be more important, because adenovirus is amuch larger virus. The Ag30-MHCs achieved almost a 2-log10

reduction of M13 within 6 h (our unpublished data). M13 is sin-gle-stranded DNA virus (35) and is �880 nm in length and 6.6 nmin diameter (36). The rate of inactivation of the filamentous bac-teriophage M13 was intermediate between those of small, round-structured viruses (MNV, �X174, and MS2) and a large virus(AdV2).

FIG 3 Antiviral activities of Ag30-MHCs after exposure to various pH condi-tions. (a) Bacteriophage MS2; (b) bacteriophage �X174. First, Ag30-MHCswere exposed to acidic (pH 2.0 and 5.0) and alkaline (pH 9.0 and 12.0) condi-tions, created by adding 0.1 N HCl or 0.1 N NaOH to distilled water, in ashaking incubator (150 rpm, 10 min, 25°C). The acid or alkali was neutralized,and the target microorganisms were reacted with the treated and untreated(control, pH 7.6) Ag30-MHCs in a shaking incubator (150 rpm, 1 h, 25°C).Values are expressed as means � standard deviations (SD) from three inde-pendent experiments. Asterisks indicate significantly different values (P 0.05by Kruskal-Wallis one-way ANOVA with Dunnett’s test).

FIG 4 Inactivation of bacteriophages MS2 (a) and �X174 (b) by Ag30-MHCsexposed to tap and surface water. First, Ag30-MHCs were exposed to tap wateror to surface water samples (F, filtered; NF, nonfiltered) from the Han River ina shaking incubator (150 rpm, 10 min, 25°C). Then, the bacteriophages wereadded to the water samples in a shaking incubator (150 rpm, 1 h, 25°C). Valuesare expressed as means � standard deviations (SD) from three independentexperiments. Asterisks indicate significantly different values (P 0.05 byKruskal-Wallis one-way ANOVA with Dunnett’s test).

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In the present study, the inactivation kinetics of MNV weresignificantly different in the plaque assay and the RT-PCR assay(Fig. 2b and c). Molecular assays such as RT-PCR are known tosignificantly underestimate the inactivation rates of viruses (25,28), as was found in our study. Because AgNP-MHCs interactprimarily with the viral surface, they are more likely to damageviral coat proteins rather than viral nucleic acids. Therefore, anRT-PCR assay to measure viral nucleic acid may significantly un-derestimate the inactivation of viruses. The effect of Ag� ions andthe generation of reactive oxygen species (ROS) should be respon-sible for the inactivation of the tested viruses. However, manyaspects of the antiviral mechanisms of AgNP-MHCs are still un-clear, and further research using biotechnological approaches isrequired to elucidate the mechanism of the high resistance ofAdV2.

Finally, we evaluated the robustness of the antiviral activity ofAg30-MHCs by exposing them to a wide range of pH values and totap and surface water samples before measuring their antiviralactivity (Fig. 3 and 4). Exposure to extremely low pH conditionsresulted in the physical and/or functional loss of AgNPs from theMHC (unpublished data). In the presence of nonspecific particles,Ag30-MHCs consistently displayed antiviral activity, despite a de-cline in the antiviral activity against MS2. The antiviral activity waslowest in unfiltered surface water, as an interaction between Ag30-MHCs and nonspecific particles in the highly turbid surface waterprobably caused a reduction of antiviral activity (Table 3). There-fore, field evaluations of AgNP-MHCs in various types of water(e.g., with different salt concentrations or humic acid) should beperformed in the future.

In conclusion, the new Ag composites, AgNP-MHCs, have ex-cellent antiviral capabilities against several viruses, including�X174 and MNV. AgNP-MHCs maintain strong efficacy underdifferent environmental conditions, and these particles can be eas-ily recovered using a magnet, thus reducing their potential harm-ful effects on human health and the environment. This studyshowed that the AgNP composite can be an effective antiviral invarious environmental settings, without significant ecologicalrisks.

ACKNOWLEDGMENTS

This work was supported by National Research Foundation of Korea(NRF) grants funded by the Korean government (MEST) (no. 2012-0008692 and no. 2012-0009628) and by the BK21 Plus project(22A20130012682).

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TABLE 3 Results of water quality analysis of tap and surface watersamples

ParameterDrinking waterstandarda

Value in:

Tap waterSurface water(Han River)

Total colony count 100 CFU/ml ND 4,700c

Total coliforms NDb/100 ml ND Detectedc

Escherichia coli/fecal coliforms ND/100 ml ND Detectedc

Hardness 300 mg/liter 51 61.0pH 5.8–8.5 7.3 7.4Turbidity 0.5 NTUd 0.15 6.37c

Total solids 500 mg/liter 94 87Chloride (Cl�) 250 mg/liter 11 7Chlorine residual concn 4.0 mg/liter ND 0.48Sulfate (SO4�) 200 mg/liter 9 11Ammonium nitrogen

(NH3-N)0.5 mg/liter ND 0.09

Nitrate nitrogen (NO3-N) 10 mg/liter 2.3 2.0Fluoride (F) 1.5 mg/liter ND NDLead (Pb) 0.01 mg/liter ND NDMercury (Hg) 0.001 mg/liter ND NDCadmium (Cd) 0.005 mg/liter ND NDTrihalomethanes 0.1 mg/liter 0.029 NDToluene 0.7 mg/liter 0.006 NDEthylbenzene 0.3 mg/liter 0.007 NDXylene 0.5 mg/liter 0.011 NDa Standards for drinking water quality from the Korean EPA.b ND, not detected.c The level of the contaminant exceeded drinking water quality standards.d NTU, nephelometric turbidity units.

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